Host plants and pollination regions for the long‐distance migratory noctuid moth, Hadula trifolii Hufnagel in China

Abstract Nocturnal moths are important pollinators of plants. The clover cutworm, Hadula trifolii, is a long‐distance migratory nocturnal moth. Although the larvae of H. trifolii are polyphagous pests of many cultivated crops in Asia and Europe, the plant species pollinated by the adult are unclear. Pollen species that were attached to individual migrating moths of H. trifolii were identified based on pollen morphology and DNA to determine their host plants, geographic origin, and pollination areas. The moths were collected on their seasonal migration pathway at a small island, namely Beihuang, in the center of the Bohai Sea of China during 2014 to 2018. Pollen was detected on 28.60% of the female moths and 29.02% of the male, mainly on the proboscis, rarely on compound eyes and antennae. At least 92 species of pollen from 42 plant families, mainly from Asteraceae, Amaranthaceae, and Pinaceae, distributed throughout China were found on the test moths. Migratory H. trifolii moths visited herbaceous plants more than woody plants. Pollen of Macadamina integrifolia or M. tetraphylla was found on moths early in the migratory season. These two species are distributed in Guangdong, Yunnan, and Taiwan provinces in China, indicating that migratory moths probably traveled about 2000 km from southern China to the Beihuang Island in northern China. Here, by identifying plant species using pollen, we gained a better understanding of the interactions between H. trifolii moths and a wide range of host plants in China. This work provides valuable and unique information on the geographical origin and pollination regions for H. trifolii moths.


| INTRODUC TI ON
Latitudinal migrations of billions of animals on land or through the water or air lead to great seasonal exchanges of biomass and nutrients across the Earth (Alerstam & Bäckman, 2018;Chapman et al., 2015;Dingle & Drake, 2007;Guo et al., 2020;Hu et al., 2016).
The migration of insects, the most species-rich and abundant group of macroscopic organisms on the planet, is linked to numerous ecosystem services including pollination, biological invasion, niche competition, outbreaks of agricultural and forestry pests, over large areas (Hendrix et al., 1987;Song et al., 2021;Wäckers et al., 2007;Weiner et al., 2014;Zhang et al., 2022). Tracking the movement of insects in their natural habitat is thus essential for understanding their basic biology, demography, ethology, and ecological function.
Moths are the major nocturnal pollinators of plants (Devoto et al., 2011;Lecroy et al., 2013). At least 289 species of plants from 75 families are partially or exclusively pollinated by moths belonging to 21 families (Macgregor et al., 2015). Moths visit flowers and feed on nectar and/or pollen to meet energy needs for flight and nutritional requirements for reproduction. As a result of this visitation and feeding activity, moths pick up pollen, which can be used to identify the plant species. Thus, pollen is an outstanding natural marker for mark-capture studies of insect migration and their host plants (Bryant et al., 1991;Chang et al., 2018;Guo et al., 2018;Hagler & Jackson, 2001;Lingren et al., 1993;Liu et al., 2016;Liu, Fu, et al., 2017).
Pollen identification is remarkably useful to study the movement of insects and insect-plant interactions for three reasons (Hagler & Jackson, 2001). Firstly, the hard outer wall of pollen grains is composed of sporopollenin, one of the most durable protein materials (Hagler & Jackson, 2001;Mackenzie et al., 2015). Secondly, the distinctive morphological characteristics of pollen grains enable it to be identified to the genus level Hesse et al., 2009).
Thirdly, the distribution and flowering period of most plants are also well known, which helps to determine the geographic origin of collected insects (Chang et al., 2018;Hendrix et al., 1987;Liu et al., 2016;Liu, Fu, et al., 2017).
Pollen grains can be identified to the genus or even the species level using light microscopy (LM), scanning electron microscopy (SEM), and DNA metabarcoding. Light microscopy for pollen identification is constrained by low resolution, and preparation methods often generate confusing contaminants such as insect lipids and chitins (Hagler & Jackson, 2001;Turnock et al., 1978). Although SEM allows direct observation of the attached pollen grains with more detail and higher resolution than LM, it is costlier and more timeconsuming (Bryant et al., 1991;Hagler & Jackson, 2001;Turnock et al., 1978). Since the initial introduction of DNA metabarcoding, DNA-assisted identification of pollen grains has become common for identifying biological species, insect feeding preferences, and host plant distribution, and the origin of migratory insects (Chang et al., 2018;Galliot et al., 2017;Hawkins et al., 2015;Hebert et al., 2003;Jackson & Gahr, 2019;Liu et al., 2016;Liu, Fu, et al., 2017).
In this study, we used light microscopy, scanning electron microscopy, and DNA metabarcoding to identify the pollen species attached to migratory moths of the clover cutworm, Hadula trifolii Hufnagel (synonyms: the nutmeg; Apamea inquieta; Discestra trifolii; Hadena albifusa; Scotogramma trifolii) (Lepidoptera: Noctuidae) ( Figure 1), an agricultural pest in northern China and a common species in the community of insects that migrate across the Bohai Sea (Fu, 2015;Zhang et al., 2010;Zhao et al., 1992;Zhou et al., 2020).
It is globally distributed in both subtropical and temperate regions including Asia, Europe, North Africa, and North America (Federici, 1978;He, 1997;Zhang & Yu, 2021). Its larvae are a serious agricultural threat because they feed on more than 20 cultivated crop species, including potato, beet, cabbage, sunflower, wheat, corn, cotton, apple, melon, and legumes (Cass, 1959;Ren et al., 2006;Yu & Bao, 1996;Zhang & Yu, 2021;Zhao et al., 1992). In addition, H. trifolii is a long-distance migratory insect . They migrate toward the north in prevailing southerly winds during late spring (May) and early summer (June and July) and return to the south in F I G U R E 1 Representative images of Hadula trifolii (a: young larva; b: older larva; c: pupa chamber and pupa; d: adult). All images were taken by the author of this article with Nikon D5100 (a-c) and D200 (d) prevailing northerly winds during late summer and early autumn (August to October) . However, the geographical origin and migratory paths of these migratory populations are still unknown. To better estimate the risk of these insect pests to agriculture, we need to understand the origins, ranges, and food sources.
Twenty moths (♀:♂ =1:1; or all individuals if the total captured was <20) were removed from the nylon net capture bag every morning and placed singly into 2 ml tubes and stored in a −20°C freezer until microscopic inspection.

| Pollen examination and scanning electron microscopy (SEM) preparation
Pollen is usually observed on the proboscis, antennae, compound eyes, and legs of moths (Bryant et al., 1991;Liu et al., 2016). To clear the presence of pollen, the heads of adult H. trifolii were excised and examined with an Olympus SZX16 stereomicroscope. To prevent contamination, we washed the microscope slide under sample and all forceps with ethanol before examining each new sample (Liu et al., 2016;Liu, Fu, et al., 2017). Pollen grains found on the head (i.e., proboscis, antennae, and eyes) were placed on double-sided sticky tape on aluminum stubs, sputter-coated with gold in a sputter coater and imaged with a Hitachi S-4800 or SU8010 cold field emission SEM (Hitachi High-Technologies Co.).
The PCR mixture and thermocycling conditions of Chang et al. (2018) were used with the GeneAmp PCR System 9700 thermocycler (Applied Biosystems).
The separation and purification methods of PCR amplicons were the same as those of Chang et al. (2018). The purified products were subcloned into pEASY-T3 Cloning Vector (TransGen Biotech). The inserts were then sequenced with standard M13 primers (Shanghai Sangon), and Sanger sequencing of pollen was done by the Taihe Biotechnology Co., Ltd.

| Data analyses
Differences in taxa and number of H. trifolii moths with adhering pollen per season or per year (frequency) during different migratory seasons were analyzed using a one-way analysis of variance (ANOVA), of proportional data that were first arcsine square-roottransformed to meet assumptions of normality and heteroscedasticity. Tukey's honestly significant difference (HSD) was used as a post hoc test. The annual mean frequencies of pollen deposits on male and female moths were compared for differences using Student's t test. Differences in annual percentages of pollen on male and female moths and the characteristics of pollen source plants were all compared using a chi-squared test. All statistical analyses were done in SPSS 20.0 (IBM), except for the log rank test, which was done in GraphPad Prism 8 (GraphPad Software Inc.).

| Plant hosts deduced from pollen
During the study, 1985 moths of H. trifolii were collected; 27% had pollen grains on the proboscis, 2.32% had pollen on the antennae, and 0.6% on compound eyes. For most individuals that had pollen adhering to the body (i.e., 89.16%), the pollen was from one species; the remainder had pollen from two or three species. Ninety-two pollen species from at least 42 families were discovered on the moths (Table 1,  type of pollen from 42 families were identified, and type for 14 pollen grains were unidentified. Thus, the identification success rate using a combination of pollen morphology and DNA sequences was 17.91% to species and 41.80% to genus. Using DNA sequences only, the rate was 5.97% to species and 55.22% to genus, and pollen morphology only, the rate was 4.48% to species and 31.30% to genus.

| Annual and seasonal differences in pollencarrying frequencies
For the 1985 moths of H. trifolii that were collected and observed for pollen grains, annual percentages of pollen-bearing individuals differed among years (Table 2; χ 2 = 76.700, df = 4, p < .001). For the 572 pollen grains on the test moths, 42 families, 61 genera, and 17 species were identified ( Table 2) (Table 3).
In addition, neither the number of identified pollen type, nor the frequency of pollen adherence on the moths differed among different migratory seasons during 2014 to 2018 (number of type: Figure 3a; frequency: F 2, 10 = 0.559, p = .589, Figure 3b).

| Growth forms of pollen-bearing host plants
The plant species identified for the adherent pollen grains represented a variety of growth forms: trees, shrubs, vines, and herbs.

| Area pollinated by H. trifolii
In China, H. trifolii mainly occurs in Yunnan Province and in the northwestern and northern provinces (Figure 4a). Combining the pollen identification results and distribution information for plants in China, we found that the pollination area of H. trifolii moths extends to Shanghai in the east, Xinjiang in the west, Hainan in the south, and Heilongjiang in the north during the different migration times (Figure 4b-p).
TA B L E 1 Pollen grains carried by Hadula trifolii moths and type identified by molecular and morphological analysis and the geographic distribution of the pollen source plants  (Liu et al., 2016), Mythimna separata Walker Liu, Fu, et al., 2017), Agrotis segetum Denis and Schiffermaller (Chang et al., 2018) and Helicoverpa armigera Hübner (Zhou et al., 2019). Migratory A. ipsilon and M. separata moths (Liu et al., 2016;Liu, Fu, et al., 2017) (Chang et al., 2018). Different food sources can affect the structure and the temporal dynamics of an insect population (Wäckers et al., 2007). The quality and quantity of food sources can influence insect survival, development, flight, and reproduction Wäckers et al., 2007), and the fitness of insects usually differs depending on its nectar and/or pollen sources Liu, Zhu, et al., 2017). Of course, further study is needed to explore host-plant feeding preferences of H. trifolii adults and assess the effects on population dynamics.
Although pollen identification can be used to determine the geographical origin of migratory insects, pollen can be picked up in regions far from where the pollen-contaminated insects are captured (Hagler & Jackson, 2001). For example, Hendrix et al. (1987) found that male Heliothis zea Boddie moths captured in Arkansas probably originated from southern Texas, at least 750 km away, based on pollen from the proboscis or eye area.

Pseudaletia unipuncta Haworth and A. ipsilon moths captured in Iowa and
Missouri were contaminated with exotic pollen grains from species that only grow in southern Texas, which provided evidence that these two moths probably traveled 1300-1600 km to Iowa or Missouri from within Mexico (the state of Tamaulipas) (Hendrix & Showers, 1992). Previous studies have confirmed that H. trifolii is a long-distance migratory pest Zhang et al., 2010 (Chang et al., 2018;Liu et al., 2016;Liu, Fu, et al., 2017). Understanding the geographical origin of H. trifolii can help strengthen the management and control of this pest and secure supplies of major agricultural products .
We did not find any sex-related differences in the frequency of pollen attachment, similar to findings for the migratory noctuid moths A. ipsilon, A. segetum and M. separata (Chang et al., 2018;Liu et al., 2016;Liu, Fu, et al., 2017). This result may be due to the fact that both male and female migratory noctuid moths must feed on plants to meet nutritional requirements for the development of the internal reproductive system and for energy for flight, mating, and/or oviposition and other processes (Gilbert, 1972 (Hendrix et al., 1987) and 38.4% of female and 65% of male A.
ipsilon carried pollen (Hendrix & Showers, 1992). We found that 11.11% to 37.15% of female H. trifolii moths and 14.66% to 35.77% of the male moths carried pollen, similar to our findings for migratory A. segetum and H. armigera moths captured in Beihuang Island (Chang et al., 2018;Zhou et al., 2019). In general, pollen prevalence on A. segetum, H. armigera and H. trifolii was obviously lower than on A. ipsilon and H. zea moths. Diverse elements, involving plant phenology, nectar viscosity, pollen grain characteristics, migratory route, and antennae or mouthpart structure of insects can affect flower visitation patterns and associated variability in the frequency that an insect carries pollen (Krenn, 2010;Liu et al., 2016;Tudor et al., 2004). In addition, moth collection methods may also alter the detection rate of pollen adherence. Migratory noctuid moths captured on Beihuang Island were collected using a searchlight trap, with numerous species and individuals of insects (Guo et al., 2020). A large number of insects are collected in a bag and may be squeezed or rubbed together, which may knock off adherent pollen. However, sex pheromone traps are highly specific and capture fewer insect species than light traps; thus, pollen is less likely to be removed.
The frequency of pollen attachment to field-collected insects can also be used to infer the relative importance or contribution of the nectar plants (Chang et al., 2018;Jones & Coppedge, 1999).
We found notable seasonal differences in the families represented by pollen type on migratory H. trifolii moths, with more Pinaceae early in the migratory season, Amaranthaceae middle season, and Asteraceae late in the season. This finding was similar to that for A.
segetum (Chang et al., 2018). Plant phenology and species-specific flowering or pollen-shedding mechanism can explain seasonal vari- During their close interactions, plants and insects coevolve.
Moth abundance has decreased significantly in recent decades, and their occurrence is likely to be affected by many environmental factors including light pollution and changes in land-use and climate (Fox et al., 2011(Fox et al., , 2014Macgregor et al., 2015;Péter et al., 2020). The larvae of most lepidopteran insects are agricultural and forestry pests, while the adults (moths or butterflies) are usually pollinators of many plant species and a food source for many organisms (such as birds, bats, fishes, frogs, and spiders) (Devoto et al., 2011;Fox, 2013;Kato & Kawakita, 2017;Liu, Fu, et al., 2017). Therefore, a decrease in moth abundance will also affect the abundance of other organisms in the ecosystem. The ecological functions of agricultural pests need to be continuously explored and assessed. With regard to pollination ecology, the contribution of pollinators other than bees (e.g., beetles, flies, moths, and butterflies) have been little explored although their role in pollination processes is F I G U R E 3 Number of type (a) and frequencies (b) of pollen grains and type of host plants (c, d, and e) represented by pollen grains attached to migratory individuals of Hadula trifolii in 2014-2018. Different letters above bars in panel a or b indicate a significant difference among means (p > .05, one-way ANOVA followed by Tukey's HSD test), and double asterisks (**) in panels c-e indicates a significant difference between means (p < .01, chi-squared test) well known (Devoto et al., 2011;Galliot et al., 2017;Lecroy et al., 2013;Matsuka & Sakai, 2015;Weiss, 2001

| CON CLUS IONS
Pollen grain identification is a practical means to study pollination ecology, insect movement, and plant-insect interactions. Here, by identifying plant species using pollen, we gained a better understanding of the interactions between H. trifolii moths and a wide range of host plants in China. Our work advances the knowledge of the nutrient relationship between a long-distance migration noctuid insect and its host plants over broad geographical scales, provides valuable and unique information on the nutrition, geographical origin and pollination service of H. trifolii moths and establishes a basis for targeted control of a global agricultural pest.

ACK N OWLED G M ENTS
This work was financially supported by the National Natural

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest. The data that support the findings of this study are available in the Supplementary material of this article.